Protein Folding and Characterization

1

• Protein Folding and Characterization

Ribonuclease A, cysteines are shown as yellow
spacefilling models.
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Lecture Overview

• Protein Folding
• Methods for Separating Proteins
• Methods for Analyzing Proteins

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Protein Denaturation

• Changes in 2o, 3o, or 4o structure by
• Heat.
• Heavy metals (Hg is most common).
• pH (trichloroacetic acid)

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Protein Denaturation
Effects of Denaturation 1. Decreased
solubility. 2. greater reactivity 3. Decreased
biological activity(enzyme) 4. Nutritive value.
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Denaturation

• Definition Conformation is changed but primary
  structure is unchanged.
• Factors affect denaturation high temperature,
  pressure, chemicals, etc.
• Denatured proteins lose their bioactivity.
• Renaturation restore their native conformation
  and function.

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• Urea denatures (unfolds) a protein by
  disrupting noncovalent bonds in the protein that
  stabilize the three-dimensional structure.
• In the unfolded protein, hydrophobic side
  chains are exposed to the aqueous environment.

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Refolding of ribonuclease restores all of its
enzymatic activity

• Denatured ribonuclease refolds into its
  native structure when urea and dithiothreitol
  (DTT) are removed.

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Leventhals paradox
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• Suppose that each conformation is converted
  into another conformation in the shortest
  possible time (10-15 seconds). Then the time
  required to sample all possible conformations of
  the polypeptide chain will be

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• Since proteins generally fold within a few
  seconds, they obviously arent trying out every
  possible conformation.
• Leventhals paradox how do proteins find the
  correct conformation if it takes 1077 years to
  sort through all of the possible conformations.
• There must be some order to the protein
  folding process.

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Protein unfolding occurs through a molten
globule intermediate
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Chaperones catalyze protein folding
13

• Since protein folding is such an important
  process, one might expect that there are enzymes
  that catalyze the rate at which protein folding
  occurs. This is indeed the case.
• Proteins that catalyze protein folding are
  known as chaperones, such as HSP70.

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Protein disulfide isomerase catalyzes internal
disulfide bond exchange
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Prolyl isomerase catalyzes the isomerization of
cis and trans prolines
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Proteins separation and analysis
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Dialysis separates proteins based on size

• The driving force for dialysis is
  diffusion-from high concentration to low
  concentration.

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Centrifugation separates proteins based on mass,
size, and shape
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• The sedimentation velocity of a particle
  depends on its mass, shape, density, and the
  density of the solution that it is in.
• Particles with large sedimentation
  coefficients sediment more rapidly than particles
  with small sedimentation coefficients.

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Gel filtration chromatography separates proteins
based on size

• The chromatography column is filled with
  small, porous beads. Small molecules can enter
  the spaces in the beads, while large molecules
  are excluded from the beads. Since large
  molecules cant enter the beads, they flow
  through the column more rapidly than small
  molecules.

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Isoelectric focusing separates proteins based on
charge
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Proteins can be separated based on their
isoelectric point

• The isoelectric point (pI) is the pH at which
  the protein has an equal number of positive and
  negative charges

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SDS-PAGE separates proteins based on their
molecular weight

• In SDS-polyacrylamide gel electrophoresis
  (SDS-PAGE), the proteins are coated with the
  detergent SDS (sodium dodecyl sulfate). SDS is
  negatively charged,.

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The rate at which a protein migrates in SDS-PAGE
is proportional to the log of its molecular
weight
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Protein concentration determination
1.Kjeldahl Method Nitrogen ? 6.25
Protein 2. Ultraviolet Method
Chromophoric side chains of aromatic AA
(Tyrosine,Tryptophan) have Absorption at 280 nm.
Non-destructive means to determine protein.
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Amino acid analysis gives the composition of
proteins

• Acid hydrolysis results in cleavage of the
  peptide bonds in a protein. Acid hydrolysis of
  the insulin A chain is shown.

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Amino acids can be separated by HPLC
The relative numbers of each amino acid in the
hydrolyzed protein can be quantified by HPLC.
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Proteins can be separated into peptides by
proteolytic digestion

• The protease trypsin cleaves after lysines
  and arginines. The trypsin cleavage of
  ribonuclease A is shown.

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Separation of peptides Peptide fingerprinting
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Analysis of Proteins Peptide sequencing
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Labeling reaction
Amino acid release
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• Edman degradation method
• the carboxyl-terminus of the peptide is
  attached to a solid support. In each step, only
  the amino-terminal AA is labeled by PITC. As a
  result, only the amino-terminal peptide bond is
  cleaved in the reaction. After the labeling
  reaction, excess PITC is washed off and the
  peptide is incubated with mild acid (peptide bond
  cleavage is catalyzed by mild acid). Release of
  the first amino acid from the peptide produces a
  new amino acid at the amino-terminus of the
  peptide, which can now react with PITC. The
  labeling/cleavage procedure is repeated until all
  of the amino acids in the peptide have been
  sequenced.